CD4+ T follicular helper cells (Tfh) cells are required to provide help for antigen-specific germinal center B cell responses, thereby promoting the affinity maturation of antibodies and the generation of long-lived plasma cell and memory B cells. Therefore, understanding how Tfh cells develop and exert their effector function is of great interest for efforts to promote antibody-mediated protection against infectious diseases through vaccination. Following acute viral infection, nave CD4 T cells specific for viral antigen undergo dramatic changes, including robust proliferation, and differentiation to the distinct T helper 1 (Th1) and Tfh cell lineages. Following clearance of virus, subsets of memory Th1 and Tfh cells are maintained, with the ability to recall their lineage-specific functions upon re-exposure to antigen. In Aim1 of this research project, we will investigate how CD4 Tfh effector and memory cells differentiate during viral infection versus immunization with a protein antigen. We will determine whether these different types of immune reactions alter the function and lineage stability of Tfh and Th1 memory cells upon antigenic rechallenge. We will also determine whether repeated boosting with antigen or heterologous infections reinforces Tfh and Th1 lineage commitment, or reprograms these cells to acquire new phenotypic and functional traits. In Aim 2 of this proposal, we will investigate how epigenetic mechanisms mediated by changes in DNA methylation regulate the ability of lineage-committed Tfh and Th1 memory cells to remember their previously programmed effector gene expression, and repress transcription of genes relevant to other T helper lineages. This will be done using genome-wide methylation analysis, validation of candidate loci that are differentially methylated, and functional experiments to determine whether cells re-express unmethylated (poised) and methylated (repressed) loci upon reactivation of memory Tfh and Th1 cells. Finally, we will explore whether new DNA methylation programs are acquired during Tfh and Th1 cell differentiation, and whether this de novo methylation influences the lineage-commitment of memory CD4 T cells. Together, these studies will provide novel understanding into the programming of virus infection- and immunization-induced memory CD4 T cells, which in turn would allow us to direct vaccine- mediated responses toward improved lasting immune protection against infectious diseases.